Many members of the tumor necrosis factor (TNF) family of ligands and their corresponding receptors regulate growth of normal cells by inducing apoptosis or enhancing cell survival and proliferation. It is this balance between apoptotic signals and survival and proliferation signals that maintains normal cellular homeostasis. At least 26 TNF family receptors and 18 TNF family ligands have been identified to date. The biologically active forms of both the receptors and ligands are self-assembled protein trimers. Transmembrane and soluble forms of both the receptors and ligands have been identified. Though the intracellular domains of the receptors share no sequence homology, their extracellular domains comprise 40-amino-acid, cysteine-rich repeats. Their cytoplasmic tails signal by interacting with two major groups of intracellular proteins: TNF receptor-associated factors (TRAFs) and death domain (DD)-containing proteins. Interaction between at least six human TRAFs and TRAF-binding sites on the cytoplasmic tail of some of these receptors initiates several signaling pathways, including AKT (the serine/threonine kinase referred to as protein kinase B or PKB), nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK). See, for example, the review by Younes and Kadin (2003) J. Clin. Oncol. 18:3526-3534.
The TNF family receptor member CD40 is a 50-55 kDa cell-surface antigen present on the surface of both normal and neoplastic human B cells, dendritic cells, monocytes, macrophages, CD8+ T cells, endothelial cells, monocytic and epithelial cells, and many solid tumors, including lung, breast, ovary, urinary bladder, and colon cancers. Binding of the CD40 ligand (CD40L) to the CD40 antigen on the B cell membrane provides a positive costimulatory signal that stimulates B cell activation and proliferation, resulting in B cell maturation into a plasma cell that secretes high levels of soluble immunoglobulin. CD40 activates TRAF-2, -3, -5, and -6, which upregulate diverse signaling pathways following engagement of CD40 with CD40L (either membrane-bound CD40L or soluble CD40L), including extracellular signal-regulated kinase (ERK), c-jun amino terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), AKT, and NF-κB (see, for example, Younes and Carbone (1999) Int. J. Biol. Markers 14:135-143; van Kooten and Banchereau (2000) J. Leukoc. Biol. 67:2-17).
Malignant B cells from tumor types of B-cell lineage express CD40 and appear to depend on CD40 signaling for survival and proliferation. Transformed cells from patients with low- and high-grade B-cell lymphomas, B-cell acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia, Walsdenstrom's Macroglobulinemia, and Hodgkin's disease express CD40. CD40 expression is also detected in acute myeloblastic leukemia and 50% of AIDS-related lymphomas.
A number of carcinomas and sarcomas also exhibit high levels of CD40 expression, though the role of CD40 signaling in these cancer cells is less well understood. CD40-expressing carcinomas include urinary bladder carcinoma (Paulie et al. (1989) J. Immunol. 142:590-595; Braesch-Andersen et al. (1989) J. Immunol. 142:562-567), breast carcinoma (Hirano et al. (1999) Blood 93:2999-3007; Wingett et al. (1998) Breast Cancer Res. Treat. 50:27-36); prostate cancer (Rokhlin et al. (1997) Cancer Res. 57:1758-1768), renal cell carcinoma (Kluth et al. (1997) Cancer Res. 57:891-899), undifferentiated nasopharyngeal carcinoma (UNPC) (Agathanggelou et al. (1995) Am. J. Pathol. 147:1152-1160), squamous cell carcinoma (SCC) (Amo et al. (2000) Eur. J. Dermatol. 10:438-442; Posner et al. (1999) Clin. Cancer Res. 5:2261-2270), thyroid papillary carcinoma (Smith et al. (1999) Thyroid 9:749-755), cutaneous malignant melanoma (van den Oord et al. (1996) Am. J. Pathol. 149:1953-1961), gastric carcinoma (Yamaguchi et al. (2003) Int. J. Oncol. 23(6):1697-702), and liver carcinoma (see, for example, Sugimoto et al. (1999) Hepatology 30(4):920-26, discussing human hepatocellular carcinoma). For CD40-expressing sarcomas, see, for example, Lollini et al. (1998) Clin. Cancer Res. 4(8):1843-849, discussing human osteosarcoma and Ewing's sarcoma.
CD40 signaling protects immature B-cells and B-cell lymphomas from apoptosis induced by IgM or Fas (see, for example, Wang et al. (1995) J. Immunol. 155:3722-3725). Mantle cell lymphoma cells express a high level of CD40, and the addition of exogenous CD40 ligand was shown to enhance their survival and rescue them from fludarabine-induced apoptosis (Clodi et al. (1998) Brit. J. Haematol. 103:217-219). The role of CD40 signaling in malignant B cell survival and proliferation renders the CD40 antigen a potential target for anti-cancer therapy. Indeed, antagonist anti-CD40 antibodies inhibit proliferation and/or differentiation of malignant human B cells in vitro (see, for example, U.S. Patent Application Publication No. 20040109857). Further, murine models of aggressive human lymphomas have demonstrated the in vivo efficacy of anti-CD40 antibodies in promoting animal survival. See, for example, Funakoshi et al. (1994) Blood 83:2787-2794; Tutt et al. (1998) J. Immunol. 161:3176-3185; and Szocinski et al. (2002) Blood 100: 217-223.
The CD40 ligand (CD40L), also known as CD154, is a 32-33 kDa transmembrane protein that also exists in two smaller biologically active soluble forms, 18 kDa and 31 kDa, respectively (Graf et al. (1995) Eur. J. Immunol. 25:1749-1754; Mazzei et al. (1995) J. Biol. Chem. 270:7025-7028; Pietravalle et al. (1996) J. Biol. Chem. 271:5965-5967). CD40L is expressed on activated, but not resting, CD4+ T-helper cells (Lane et al. (1992) Eur. J. Immunol. 22:2573-2578; Spriggs et al. (1992) J. Exp. Med. 176:1543-1550; and Roy et al. (1993) J. Immunol. 151:1-14). Both CD40 and CD40L have been cloned and characterized (Stamenkovi et al. (1989) EMBO J. 8:1403-1410; Armitage et al. (1992) Nature 357:80-82; Lederman et al. (1992) J. Exp. Med. 175:1091-1101; and Hollenbaugh et al. (1992) EMBO J. 11:4313-4321). See also U.S. Pat. No. 5,945,513, describing human CD40L. Cells transfected with the CD40L gene and expressing the CD40L protein on their surface can trigger B-cell proliferation, and together with other stimulatory signals, can induce antibody production (Armitage et al. (1992) supra; and U.S. Pat. No. 5,945,513). Patients with lymphoid malignancies, autoimmune disease, cardiovascular disease, and essential thrombocythemia have elevated serum levels of soluble CD40L (sCD40L) that are not seen in healthy subjects. Constitutive expression of CD40L has been observed in a subset of patients with several B-cell lymphoid malignancies, including mantle-cell lymphoma, follicular lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and HIV-infected B-cell lymphoma. See, for example, Clodi et al. (1998) Br. J. Haematol. 103:270-275; Schattner et al. (1998) Blood 91:2689-2697; Moses et al. (1997) Nat. Med. 3:1242-1249; Trentin et al. (1997) Cancer Res. 57:4940-4947; and Pham et al. (2002) Immunity 16:37-50). CD40L may play an important role in the cell contact-dependent interaction of CD40-expressing tumor B-cells within the neoplastic follicles or CD40-expressing Reed-Sternberg cells in Hodgkin's disease areas (Carbone et al. (1995) Am. J. Pathol. 147:912-922). However, the mechanism of CD40L-mediated CD40 signaling leading to survival versus cell death responses of malignant B-cells is not completely known. For example, in follicular lymphoma cells, down-regulation of apoptosis-inducing TRAIL molecule (APO-2L) (Ribeiro et al. (1998) British J. Haematol. 103:684-689) and over expression of bcl-2, and in the case of B-CLL, down-regulation of CD95 (Fas/APO-1) (Laytragoon-Lewin et al. (1998) Eur. J. Haematol. 61:266-271) have been proposed as mechanisms of survival. In contrast, evidence in follicular lymphoma indicates that CD40 activation leads to up-regulation of TNF (Worm et al. (1994) International Immunol. 6:1883-1890) and CD95 molecules (Plumas et al. (1998) Blood 91:2875-2885).
Human anti-CD40 monoclonal antibodies and a number of uses thereof are disclosed in co-owned patent applications published as WO 2005/044854, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044855, WO 2005/044307, and WO 2005/044294. Those applications specifically disclose a human IgG1 anti-CD40 monoclonal antibody, designated as CHIR-12.12 therein, which was generated by immunization of transgenic mice bearing the human IgG1 heavy chain locus and the human κ light chain locus (XenoMouse® technology; Abgenix, Calif.).
As shown by FACS analysis, CHIR-12.12 binds specifically to human CD40 and can prevent CD40-ligand (CD40L) binding. CHIR-12.12 can compete off CD40L pre-bound to cell surface CD40. The CHIR-12.12 monoclonal antibody is a strong antagonist and inhibits in vitro CD40L-mediated proliferation of normal and malignant B cells. The CHIR-12.12 monoclonal antibody directly inhibits survival and signaling pathways mediated by CD40L in normal human B-lymphocytes. In vitro, CHIR-12.12 kills primary cancer cells from NHL patients by ADCC. Dose-dependent anti-tumor activity was seen in a xenograft human lymphoma model. CHIR-12.12 is currently in Phase I trials for B-cell malignancies.
CD20 is a cell-surface antigen expressed early in B cell differentiation and remains on the cell surface throughout B cell development. CD20 is involved in B cell activation, is expressed at very high levels on neoplastic B cells, and is a clinically recognised therapeutic target (see, for example, Hooijberg et al. (1995) Cancer Research 55: 2627). Antibodies targeting CD20, such as rituximab (Rituxan®), have been approved by the U.S. Food and Drug Administration for the treatment of non-Hodgkin's lymphoma (see, for example, Boye et al. (2003) Ann. Oncol. 14:520). Rituxan® has been shown to be an effective treatment for low-, intermediate-, and high-grade non-Hodgkin's lymphoma (NHL) and active in other B-cell malignancies (see for example, Maloney et al. (1994) Blood 84:2457-2466), McLaughlin et al. (1998) J. Clin. Oncol. 16:2825-2833, Maloney et al. (1997) Blood 90:2188-2195, Hainsworth et al. (2000) Blood 95:3052-3056, Colombat et al. (2001) Blood 97:101-106, Coiffer et al. (1998) Blood 92:1927-1932), Foran et al. (2000) J. Clin. Oncol. 18:317-324, Anderson et al. (1997) Biochem. Soc. Trans. 25:705-708, or Vose et al. (1999) Ann. Oncol. 10:58a).
Though the exact mechanism of action is not known, evidence indicates that the anti-lymphoma effects of Rituxan® are in part due to complement-mediated cytotoxicity (CMC), antibody-dependent cell-mediated cytotoxicity (ADCC), inhibition of cell proliferation, and finally direct induction of apoptosis. ADCC is a major mechanism of action for many marketed and investigational monoclonal antibodies. Some patients, however, become resistant to treatment with Rituxan® (see Witzig et al. (2002) J. Clin. Oncol. 20:3262, Grillo-Lopez et al. (1998) J. Clin. Oncol. 16:2825, or Jazirehi et al. (2003) Mol. Cancer. Ther. 2:1183-1193). For example, some patients lose CD20 expression on malignant B cells after anti-CD20 antibody therapy (Davis et al. (1999) Clin. Cancer Res. 5:611). Furthermore, 30% to 50% of patients with low-grade NHL exhibit no clinical response to this monoclonal antibody (Hainsworth et al. (2000) Blood 95:3052-3056; Colombat et al. (2001) Blood 97:101-106). The clinical activity of rituximab in NHL has also been shown to be correlated with the patient's FcγRIIIa genotype. Patients with the FcγRIIIa 158aa polymorphism of V/V or V/F are more responsive to rituximab than those with F/F (for example, see Cartron et al. (2002) Blood 99(3):754-758 or Dall'Ozzo et al. Cancer Res. (2004) 64:4664-4669). For patients developing resistance to this monoclonal antibody, or having a B-cell lymphoma that is resistant to initial therapy with this antibody, alternative forms of therapeutic intervention are needed.
There is thus a continuing need for new therapeutic agents and new therapeutic strategies for cancers and pre-malignant conditions. In particular, there is a need for new therapeutic strategies for treatment of patients who are homozygous or heterozygous for FcγRIIIa-158F and are refractory to treatment with anti-CD20 antibodies, such as rituximab (Rituxan®). Moreover, an antibody that can kill malignant cells without needing a conjugate will result in a drug that is cheaper to make and could have fewer side effects.